The ability to differentiate true malaria relapse from reinfection has significant practical implications in areas endemic for Plasmodium vivax malaria. For example, if the majority of infections in a given village are relapses instead of new infections then mosquito spraying efforts can be concentrated during the period of intense transmission and scarce resources will be targeted towards effective measures of prevention. Morphologically there is no difference between the parasites seen during the original parasitemia and during relapse. Accordingly, we have designed molecular markers using conserved sequences of the circumsporozoite protein (CSP) and the merozoite surface protein (MSP) genes to determine if these parasites are different at the molecular level. Using the polymerase chain reaction (PCR) we were able to amplify DNA from blood samples taken from rhesus monkeys infected with P. cynomolgi. We next sequenced the amplicons from both relapses and initial infections and compared the sequences. We found that the parasites seen in the initial bloodstream infection with P. cynomolgi in rhesus monkey are identical at the molecular level to those parasites found in the 1st, 2nd and 3rd relapse. This finding in the monkey model has enabled us to take this technique to the field and examine blood samples from people in a Plasmodium vivax malaria-endemic area of Colombia where we were able to differentiate relapse from reinfection using these markers.